Post translational modification of proteins by covalent attachment of carbohydrate moieties is common among eukaryotes, but by comparison, relatively rare in bacteria. Currently, two virulence factors of P. aeruginosa, pili and the a-type flagellin as well as a protein responsible for phospholipase transport, are reported to be glycosylated. The long term goal of this application is therefore to understand the role of glycosylation in virulence of P. aeruginosa. The specific aims of this application are intended to study the role of glycosylation of the flagellum in P. aeruginosa infections, the molecular bias of glycosylation system. We will construct chimeric flagellins that will consist of glycosylated or nonglycosylated regions in the same genetic background. These mutants will be compared to the glycosylated a-type parent strain for virulence in animal models of infection. Next, flagellins with higher degrees of glycosylation will be compared to the PAK parent strain in an animal model to examine whether there is a relationship between virulence and degree of glycosylation. A "glycosylation island" that is present among P. aeruginosa flagellar genes will be examined in detail, to ascertain the roles of these genes in glycosylation. Pseudomonas homologues of genes involved in flagellar glycosylation in other bacteria will be mutagenized and examined for any for any effects on flagellin glycosylation, general protein glycosylation and LPS structure. There may also be accessory genes involved, therefore transposon mutagenesis of the a-type strain will be don't to find these. Lastly, site directed mutagenesis will be done on the potential glycosylation sites of the PAK flagellin to ascertain where glycosylation occurs and such mutants will be used to unambiguously provide evidence for a role of glycosylation in virulence. The results of these studies should provide information to assess the mechanism and role of glycosylation of the flagellum of P. aeruginosa in virulence and provide information about the relationship of flagellum glycosylation, general protein glycosylation and LPS. The existence of a central gene product involved in these could prove to be of great significance in controlling virulence.